Cellular transformation during the development of cancer involves multiple alterations in the normal pattern of cell growth regulation. Primary events in the process of carcinogenesis involve the activation of oncogene function by some means (e.g., amplification, mutation, chromosomal rearrangement), and in many cases the removal of anti-oncogene function. In the most malignant and untreatable tumors, normal restraints on cell growth are completely lost as transformed cells escape from their primary sites and metastasize to other locations in the body. One reason for the enhanced growth and invasive properties of some tumors may be the acquisition of increasing numbers of mutations in oncogenes, with cumulative effect (Bear et al., Proc. Natl. Acad. Sci. USA 86:7495-7499, 1989). Alternatively, insofar as oncogenes function through the normal cellular signalling pathways required for organismal growth and oncogenes function through the normal cellular signalling pathways required for organismal growth and cellular function (reviewed in McCormick, Nature 363:15-16, 1993), additional events corresponding to mutations or deregulation in the oncogenic signalling pathways may also contribute to tumor malignancy (Gilks et al., Mol. Cell Biol. 13:1759-1768, 1993), even though mutations in the signalling pathways alone may not cause cancer.
Several discrete classes of proteins are known to be involved in conferring the different types of changes in cell division properties and morphology associated with transformation. These changes can be summarized as, first, the promotion of continuous cell cycling (immortalization); second, the loss of responsiveness to growth inhibitory signals and cell apoptotic signals; and third, the morphological restructuring of cells to enhance invasive properties.
Of these varied mechanisms of oncogene action, the role of control of cell morphology is one of the least understood. Work using non-transformed mammalian cells in culture has demonstrated that simply altering the shape of a cell can profoundly alter its pattern of response to growth signals (DiPersio et al., Mol. Cell Biol. 11:4405-4414, 1991), implying that control of cell shape may actually be causative of, rather than correlative to, cell transformation. For example, mutation of the antioncogene NF2 leads to development of nervous system tumors. Higher eucaryotic proteins involved in promoting aberrant morphological changes related to cancer may mediate additional functions in normal cells that are not obviously related to the role they play in cancer progression, complicating their identification and characterization. Identification and characterization of such genes and their encoded proteins would be beneficial for the development of therapeutic strategies in the treatment of malignancies.
Recent evidence suggests that certain key proteins involved in control of cellular morphology contain conserved domains referred to as SH2 and SH3 domains. These domains consist of non-catalytic stretches of approximately 50 amino acids (SH3) and 100 amino acids (SH2, also referred to as the "Src homology domain"). SH2/SH3 domains are found in cytoskeletal components, such as actin, and are also found in signalling proteins such as Abl. The interaction of these proteins may play a critical role in organizing cytoskeleton-membrane attachments.
Besides the numerous SH2/SH3 containing molecules with known catalytic or functional domains, there are several signalling molecules, called "adapter proteins," which are so small that no conserved domains seem to exist except SH2 and SH3 domains. Oncoproteins such as Nck, Grb2/Ash/SEM5 and Crk are representatives of this family. The SH2 regions of these oncoproteins bind specific phosphotyrosine-containing proteins by recognizing a phosphotyrosine in the context of several adjacent amino acids. Following recognition and binding, specific signals are transduced in a phosphorylation dependent manner.
As another example, P47v-Crk (CrK) is a transforming gene from avian sarcoma virus isolate CT10. This protein contains one SH2 and one SH3 domain, and induces an elevation of tyrosine phosphorylation on a variety of downstream targets. One of these targets, p130cas, is tightly associated with v-Crk. The SH2 domain of v-Crk is required for this association and subsequent cellular transformation. P130cas is also a substrate for Src mediated phosphorylation. Judging from its structure, p130cas may function as a "signal assembler" of Src family kinases and several cellular SH2-containing proteins. These proteins bind to the SH2 binding domain of p130cas, which is believed to induce a conformational change leading to the activation in inactivation of downstream signals, modulated by multiple domains of the protein.
Another oncogene, Ras, is a member of a large evolutionarily conserved superfamily of small GTP-binding proteins responsible for coordinating specific growth factor signals with specific changes in cell shape, including the development of stress fibers and membrane ruffles (Ridley and Hall, Cell 70:389-399, 1992; Ridley et al., Cell 70:401-410,1992). A rapidly growing family of oncoproteins, including Vav, Bcr, Ect-2, and Dbl, has been found to be involved in a variety of different tumors (Eva and Aaronson, Nature 316:273-275, 1985; Ron et al., EMBO J. 7:2465-2473, 1988; Adams et al., Oncogene 7:611-618, 1992; Miki et al., Nature 362:462-465, 1993). Proteins of this family have been shown to interact with Ras/Rac/Rho family members, and possess sequence characteristics that suggest they too directly associate with and modulate organization of the cytoskeleton.
In view of the significant relationship between signalling or "adapter" proteins, altered cellular morphology and the development of cancer, it would be of clear benefit to identify and isolate such proteins (or genes encoding them) for the purpose of developing diagnostic/therapeutic agents for the treatment of cancer. It is an object of the present invention to provide a purified nucleic acid molecule of mammalian origin that encodes a signal mediator protein (SMP) involved in the signalling cascade related to morphological cellular changes, and therefrom provide isolated and purified protein. Such a gene, when expressed in model systems, such as yeast, will provide utility as a research tool for identifying genes encoding interacting proteins in the signalling cascade thereby facilitating the elucidation of the gene may also be used diagnostically to identify related genes, and therapeutically in gene augmentation or replacement treatments. It is a further object of the present invention to provide derivatives of the SMP-encoding nucleic acid, such as various oligonucleotides and nucleic acid fragments for use as probes or reagents to analyze the expression of genes encoding the proteins. It is a further object of the invention to provide the signal mediator protein in purified form, and to provide antibodies immunologically specific for the signal mediator protein for the purpose of identifying and quantitating this mediator in selected cells and tissues.